Kiesenhofer crossed the line more than a minute ahead of superstar Dutch rider Annemiek Van Vleuten who celebrated at the finish line as if she had won, not realising she had been pipped by her Austrian rival.
Italian Elisa Longo Borghini finished in third, while Tiffany Cromwell was the first Australian to cross the line in 26th, 2.56 minutes off the leader.
Australian teammate Sarah Gigante finished 8.23 minutes behind Kiesenhoder in 40th while Grace Brown came home in 47th. Amanda Spratt did not make it to the finish having abandoned the race.
It was an incredible win for Kiesenhofer who attacked from the opening stages of the 137km race and delivered a brilliantly executed display of solo racing so much so that her pursuers were unaware she was ahead.
The 30-year-old, who has not held a professional contract since 2017 and trains without a coach, was flooded with emotion after what has been called one of the biggest upsets in Olympic road race history.
“It feels incredible,” a stunned Kiesenhofer said.
“I couldn’t believe it. Even when I crossed the line, it was like, ‘Is it done now? Do I have to continue riding?’ Incredible.
“I was just trying to get to the line. My legs were completely empty. I have never emptied myself so much in my whole life. I could hardly pedal anymore. It felt like there was zero energy in my legs.”
Jubilation quickly turned to disappointment for Van Vleuten after the race when she was told she had finished second to the Austrian.
The 38-year-old revealed a lack of communication on the road led to confusion with riders unable to use race radios during the 137km race from Musashinonomori Park to the Fuji International Speedway.
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"It summarises it quite well if after the finish we are asking each other who had won, and what the time gaps were," Van Vleuten said post-race.
"We didn’t know. We heard 45 seconds with 10km to go. It showed that there was a lot of confusion and not only with me. It was in the Dutch team but also the other [nations].
“In the most important race, you’re not allowed to ride with communication, which we usually do. It should make the race more interesting but it made the race more confusing."
It was a feeling shared by Van Vleuten’s superstar teammate Anna van der Breggen.
"I didn't know there was still a rider ahead," she said.
"I thought we were riding for gold."
The Dutch team of van Vleuten, van der Breggen, Marianne Vos and Demi Vollering, arguably one of the strongest national teams ever assembled, have been criticised for their tactics, only gathering together in a concerted effort to chase in the final 10km.
While they managed to catch the remnants of the break with five kilometres to go, they did not account for Kiesenhofer holding on for the historic win.
It was Austria's first gold medal since 2004 and their first in cycling since 1896.
“It’s just so incredible,” Kiesenhofer said.
“I have really sacrificed so much for today. I wasn’t expecting to finish it off like that. I sacrificed everything even for a top-15 place and now to get this, for the sacrifices, it’s just such a reward.”
